LOCUS       sequence-272298-        5500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used as a donor vector containing N-term 3xFLAG to clone into pSLIK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5500)
  AUTHORS   Pereira EJ, Burns JS, Lee CY, Marohl T, Calderon D, Wang L, Atkins 
            KA, Wang CC, Janes KA
  TITLE     Sporadic activation of an oxidative stress-dependent NRF2-p53 
            signaling network in breast epithelial spheroids and 
            premalignancies.
  JOURNAL   Sci Signal. 2020 Apr 14;13(627). pii: 13/627/eaba4200. doi: 
            10.1126/scisignal.aba4200.
  PUBMED    32291314
REFERENCE   2  (bases 1 to 5500)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci 
            Signal."; date: "2020-04-14"; volume: "13(627). pii: 
            13/627/eaba4200. doi"; pages: " 10.1126/scisignal.aba4200"
COMMENT     Sequence Label: pEN_TTmiRC2_3xflag_Luciferase
FEATURES             Location/Qualifiers
     source          1..5500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..315
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    80..98
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    116..134
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    152..170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    187..205
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    223..241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     288..312
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             325..390
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(457..475)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     protein_bind    complement(2073..2172)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2284..2303)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2362..2381)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2955..3488)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3677..3682,3683..3699,3700..3705))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      4362..4950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4851..4870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5115..5201
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5293..5320
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     5328..5351
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    5370..5469
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
       61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
      121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
      181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
      241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
      301 aaccgtcaga tcgccactag tatggactac aaagaccatg acggtgatta taaagatcat
      361 gacatcgatt acaaggatga cgatgacaag atggaagacg ccaaaaacat aaagaaaggc
      421 ccggcgccat tctatccgct ggaagatgga accgctggag agcaactgca taaggctatg
      481 aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtggac
      541 atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat
      601 gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg
      661 ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga catttataat
      721 gaacgtgaat tgctcaacag tatgggcatt tcgcagccta ccgtggtgtt cgtttccaaa
      781 aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca aaaaattatt
      841 atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt cgtcacatct
      901 catctacctc ccggttttaa tgaatacgat tttgtgccag agtccttcga tagggacaag
      961 acaattgcac tgatcatgaa ctcctctgga tctactggtc tgcctaaagg tgtcgctctg
     1021 cctcatagaa ctgcctgcgt gagattctcg catgccagag atcctatttt tggcaatcaa
     1081 atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt
     1141 actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag atttgaagaa
     1201 gagctgtttc tgaggagcct tcaggattac aagattcaaa gtgcgctgct ggtgccaacc
     1261 ctattctcct tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac
     1321 gaaattgctt ctggtggcgc tcccctctct aaggaagtcg gggaagcggt tgccaagagg
     1381 ttccatctgc caggtatcag gcaaggatat gggctcactg agactacatc agctattctg
     1441 attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc attttttgaa
     1501 gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcaaag aggcgaactg
     1561 tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca atccggaagc gaccaacgcc
     1621 ttgattgaca aggatggatg gctacattct ggagacatag cttactggga cgaagacgaa
     1681 cacttcttca tcgttgaccg cctgaagtct ctgattaagt acaaaggcta tcaggtggct
     1741 cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc aggtgtcgca
     1801 ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga
     1861 aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa
     1921 aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac cggaaaactc
     1981 gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa gatctgagaa
     2041 ttggaagact aatgcgtttt cgagatatct agacccagct ttcttgtaca aagttggcat
     2101 tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa
     2161 aatcattatt tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca
     2221 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     2281 atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca
     2341 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     2401 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     2461 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     2521 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     2581 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     2641 aaaatattgt tgatgcgctg gcagtgtccc tgcgccggtt gcattcgatt cctgtttgta
     2701 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     2761 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     2821 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     2881 atttctcact tgataacctt atttttgacg aggggaaatt aggccgggaa gccgatctcg
     2941 gcttgaacga attgttaggt ggcggtactt gggtcgatat caaagtgcat cacttcttcc
     3001 cgtatgccca actttgtata gagagccact gcgggatcgt caccgtaatc tgcttgcacg
     3061 tagatcacat aagcaccaag cgcgttggcc tcatgcttga ggagattgat gagcgcggtg
     3121 gcaatgccct gcctccggtg ctcgccggag actgcgagat catagatata gatctcacta
     3181 cgcggctgct caaacttggg cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg
     3241 tcgaaggcag caagcgcgat gaatgtctta ctacggagca agttcccgag gtaatcggag
     3301 tccggctgat gttgggagta ggtggctacg tctccgaact cacgaccgaa aagatcaaga
     3361 gcagcccgca tggatttgac ttggtcaggg ccgagcctac atgtgcgaat gatgcccata
     3421 cttgagccac ctaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctg
     3481 cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac gcgcttgctg
     3541 cttggatgcc cgaggcatag actgtacaaa aaaacagtca taacaagcca tgaaaaccgc
     3601 cactgcgccg ttaccaccgc tgcgttcggt caaggttctg gaccagttgc gtgagcgcat
     3661 acgctacttg cattacagtt tacgaaccga acaggcttat gtcaactggg ttcgtgcctt
     3721 catccgtttc cacggtgtgc gtcacccggc aaccttgggc agcagcgaag tcgaggcatt
     3781 tctgtcctgg ctggcgaacg agcgcaaggt ttcggtctcc acgcatcgtc aggcattggc
     3841 ggccttgctg ttcttctacg gcaaggtgct gtgcacggat ctgccctggc ttcaggagat
     3901 cggaagacct cggccgtcgc ggcgcttgcc ggtggtgctg accccggatg aagtggttcg
     3961 catcctcggt tttctggaag gcgagcatcg tttgttcgcc caggactcta gctatagttc
     4021 tagtggttgg ctactaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     4081 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     4141 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     4201 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     4261 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttttcgttc
     4321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5041 gataaccgta ttaccgctag catggatctc ggggacgtct aactactaag cgagagtagg
     5101 gaactgccag gcatcaaata aaacgaaagg ctcagtcgga agactgggcc tttcgtttta
     5161 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     5221 acgttgtgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     5281 atcaaactaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc
     5341 ctgttagtta gttacttaag ctcgggcccc aaataatgat tttattttga ctgatagtga
     5401 cctgttcgtt gcaacaaatt gataagcaat gcttttttat aatgccaact ttgtacaaaa
     5461 aagcaggctt taaaggaacc aattcagtcg actggatccc
//
